Spo-rel

ABSTRACT

spo-rel polypeptides and DNA (RNA) encoding such spo-rel and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such spo-rel for the treatment of infection, particularly bacterial infections. Antagonists against such spo-rel and their use as a therapeutic to treat infections, particularly bacterial infections are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to the presence of spo-rel nucleic acid sequences and the polypeptides in a host. Also disclosed are diagnostic assays for detecting polynucleotides encoding the relA/spoT gene family and for detecting the polypeptide in a host.

This application is a divisional of U.S. application Ser. No.08/788,892, filed Jan. 22, 1997 now U.S. Pat. No. 5,989,864, whichclaims benefit of provisional application No. 60/030,151, filed Nov. 1,1996 and provisional application No. 60/029,329, filed Oct. 29, 1996.

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of these polynucleotides andpolypeptides; processes for making these polynucleotides and thesepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of these polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of the relA/spoT gene family,hereinafter referred to as “spo-rel”.

BACKGROUND OF THE INVENTION

The Staphylococci make up a medically important genera of microbes. Theyare known to produce two types of disease, invasive and toxigenic.Invasive infections are characterized generally by abscess formationeffecting both skin surfaces and deep tissues. Staphylococcus aureus isthe second leading cause of bacteremia in cancer patients.Osteomyelitis, septic arthritis, septic thrombophlebitis and acutebacterial endocarditis are also relatively common. There are at leastthree clinical conditions resulting from the toxigenic properties ofStaphylococci. The manifestation of these diseases result from theactions of exotoxins as opposed to tissue invasion and bacteremia. Theseconditions include: Staphylococcal food poisoning, scalded skin syndromeand toxic shock syndrome.

This invention provides novel spo-rel, which can be used, among otherthings, for screening for pharmaceutical compounds to treatStaphylococcal infections. In E. coli relA and spoT proteins areinvolved in the stringent response to nutrient limitation and regulatethe accumulation of (p)ppGpp which is involved in the regulation of geneexpression and other cellular processes (see Cashel, M. et al. TheStringent Response. In F. C. Neidhardt et al. (eds) Escherichia coli andSalmonella: cellular and molecular biology, 2n edition, ASM Press,Washington, D.C., 1996). The spoT protein is capable of both thesynthesis and degradation of (p)ppGpp while the relA protein is capableof (p)ppGpp synthesis only. The spoT and relA proteins are homologousbeing 55.6% similar and 31.9% identical (Metzger et al., J. Biol. Chem.264:9122-9125, 1989).

The genetic organization of the streptokinase region of theStreptococcus equisimilis H46A chromosome has been defined including thesequence of a full length ORF of a relA/spoT homolog, desingated rel(Mechold, U. et al. Mol. Gen. Genet. 241:129-140, 1993). Functionalanalysis of the rel gene from Streptococcus equisimilis indicates thatthe encoded protein is similar to E. coli spoT in that it catalyzes boththe synthesis and degradation of (p)ppGpp (Mechold, U., et al., J.Bacteriol. 178:1401-1411, 1996).

Clearly, there is a need for factors, such as spo-rel of the invention,that may be used to screen compounds for antibiotic activity and whichmay also be used to determine their roles in pathogenesis of infection,dysfunction and disease. There is a need, therefore, for identificationand characterization of such factors which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptide of the present invention has amino acid sequencehomology to known guanosine 3′, 5′-bis(diphosphate) synthetase/hydrolaseprotein.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelspo-rel peptides by homology between the amino acid sequence set out inFIG. 2 [SEQ ID NO:2] and known amino acid sequences of other proteinssuch as Streptococcus equisimilis rel protein protein.

It is a further object of the invention, moreover, to providepolynucleotides that encode spo-rel polypeptides, particularlypolynucleotides that encode the polypeptide herein designated spo-rel.

In a particularly preferred embodiment of this aspect of the inventionthe polynucleotide comprises the region encoding spo-rel polypeptides inthe sequence set out in FIG. 1 [SEQ ID NO:1], or a fragment, analogue orderivative thereof.

In another particularly preferred embodiment of the present inventionthere is a novel guanosine 3′, 5′-bis(diphosphate) synthetase/hydrolaseprotein from Staphylococcus aureus comprising the amino acid sequence ofFIG. 2 [SEQ ID NO:2], or a fragment, analogue or derivative thereof.

In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the bacterial strain Staphylococcus aureus WCUH 29contained in NCIMB Deposit No. 40771.

In accordance with this aspect of the invention there are providedisolated nucleic acid molecules encoding spo-rel, particularlyStaphylococcus spo-rel, including mRNAs, cDNAs, genomic DNAs and, infurther embodiments of this aspect of the invention includebiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives, and compositions comprising same.

In accordance with another aspect of the present invention, there isprovided the use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of spo-rel andpolypeptides encoded thereby.

In accordance with this aspect of the invention there are provided novelpolypeptides of Staphylococcus referred to herein as spo-rel as well asbiologically, diagnostically, prophylactically, clinically ortherapeutically useful fragments, variants and derivatives thereof,variants and derivatives of the fragments, and analogs of the foregoing,and compositions comprising same.

Among the particularly preferred embodiments of this aspect of theinvention are variants of spo-rel polypeptide encoded by naturallyoccurring alleles of the spo-rel gene.

In a preferred embodiment of this aspect of the invention there areprovided methods for producing the aforementioned spo-rel polypeptides.

In accordance with yet another aspect of the present invention, thereare provided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia: assessing spo-rel expression; to treat diseases and infections ofthe upper respiratory tract, e.g., otitis media, bacterial tracheitis,acute epiglottitis, thyroiditis), lower respiratory, e.g. empyema, lungabscess, cardiac, e.g. infective endocarditis, gastrointestinal, e.g.secretory diarrhea, splenic abscess, retroperitoneal abscess, CNS, e.g.cerebral abscess, eye, e.g. blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal & orbital cellulitis, darcryocystitis, kidneyand urinary tract, e.g. epididymitis, intrarenal and perinephricabscess, toxic shock syndrome, skin, e.g. impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis,and bone and joint, e.g. septic arthritis, osteomyelitis; assayinggenetic variation; and administering a spo-rel polypeptide orpolynucleotide to an organism to raise an immunological response againsta bacteria, especially a bacteria of the genus Staphylococcus.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to spo-rel polynucleotide sequences.

In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against spo-rel polypeptides.

In accordance with another aspect of the present invention, there areprovided spo-rel agonists which are also preferably bacteriostatic orbacteriocidal.

In accordance with yet another aspect of the present invention, thereare provided spo-rel antagonists which are also preferablybacteriostatic or bacteriocidal.

In a further aspect of the invention there are provided compositionscomprising a spo-rel polynucleotide or a spo-rel polypeptide foradministration to a cell or to a multicellular organism.

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIGS. 1A-1F shows the polynucleotide sequence of Staphylococcus aureusspo-rel [SEQ ID NO:1].

FIGS. 2A-2B shows the amino acid sequence of Staphylococcus aureusspo-rel [SEQ ID NO:2] deduced from the polynucleotide sequence of FIG.1.

FIG. 3A and 3B show a fragment of the polynucleotide sequence ofStaphylococcus aureus spo-rel [SEQ ID NO:7].

FIG. 4 shows the amino acid fragment of Staphylococcus aureus spo-rel[SEQ ID NO:8] deduced from the polynucleotide sequence of FIG. 3A and3B.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe Examples. The explanations are provided as a convenience and are notlimitative of the invention.

spo-rel-BINDING MOLECULE, as used herein, refers to molecules or ionswhich bind or interact specifically with spo-rel polypeptides orpolynucleotides of the present invention, including, for example enzymesubstrates, cell membrane components and classical receptors. Bindingbetween polypeptides of the invention and such molecules, includingbinding or binding or interaction molecules may be exclusive topolypeptides of the invention, which is preferred, or it may be highlyspecific for polypeptides of the invention, which is also preferred, orit may be highly specific to a group of proteins that includespolypeptides of the invention, which is preferred, or it may be specificto several groups of proteins at least one of which includes apolypeptide of the invention. Binding molecules also include antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

GENETIC ELEMENT generally means a polynucleotide comprising a regionthat encodes a polypeptide or a polynucleotide region that regulatesreplication, transcription or translation or other processes importantto expression of the polypeptide in a host cell, or a polynucleotidecomprising both a region that encodes a polypeptide and a regionoperably linked thereto that regulates expression. Genetic elements maybe comprised within a vector that replicates as an episomal element;that is, as a molecule physically independent of the host cell genome.They may be comprised within plasmids. Genetic elements also may becomprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

HOST CELL is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

IDENTITY or SIMILARITY, as known in the art, are relationships betweentwo or more polypeptide sequences or two or more polynucleotidesequences, as the case may be, as determined by comparing the sequences.In the art, identity also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated (Computational MolecularBiology, Lesk, A. M., ed., Oxford University Press, New York, 1988;Biocomputing: Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity and similarity between two polynucleotideor two polypeptide sequences, both terms are well known to skilledartisans (Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonlyemployed to determine identity or similarity between sequences include,but are not limited to those disclosed in Carillo, H., and Lipman, D.,SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determineidentity are designed to give the largest match between the sequencestested. Methods to determine identity and similarity are codified incomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F.et al., J. Molec. Biol. 215: 403 (1990)).

ISOLATED means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide or a polypeptide naturally present in a living organismin its natural state is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated”, as the term is employed herein. As part of or followingisolation, such polynucleotides can be joined to other polynucleotides,such as DNAs, for mutagenesis, to form fusion proteins, and forpropagation or expression in a host, for instance. The isolatedpolynucleotides, alone or joined to other polynucleotides such asvectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides or polypeptides, for example, into cells, compositionsor solutions for chemical or enzymatic reactions, for instance, whichare not naturally occurring compositions, and, therein remain isolatedpolynucleotides or polypeptides within the meaning of that term as it isemployed herein.

POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. Polynucleotidesembraces short polynucleotides often referred to as oligonucleotide(s).

POLYPEPTIDES, as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types. It will be appreciated that polypeptides often contain aminoacids other than the 20 amino acids commonly referred to as the 20naturally occurring amino acids, and that many amino acids, includingthe terminal amino acids, may be modified in a given polypeptide, eitherby natural processes, such as processing and other post-translationalmodifications, but also by chemical modification techniques which arewell known to the art. Even the common modifications that occurnaturally in polypeptides are too numerous to list exhaustively here,but they are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature, and they arewell known to those of skill in the art.

Among the known modifications which may be present in polypeptides ofthe present are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. Several particularly common modifications,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, forinstance, are described in most basic texts, such as, for instancePROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York (1993). Many detailed reviews areavailable on this subject, such as, for example, those provided by Wold,F., Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, as is well known and as notedabove, that polypeptides are not always entirely linear. For instance,polypeptides may be generally as a result of posttranslational events,including natural processing event and events brought about by humanmanipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli or other cells, priorto proteolytic processing, almost invariably will be N-formylmethionine.During post-translational modification of the peptide, a methionineresidue at the NH₂-terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionineless amino terminal variants of the protein of the invention.The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as, forexample, E. coli. Accordingly, when glycosylation is desired, apolypeptide should be expressed in a glycosylating host, generally aeukaryotic cell. Insect cell often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to express efficiently mammalianproteins having native patterns of glycosylation, inter alia. Similarconsiderations apply to other modifications. It will be appreciated thatthe same type of modification may be present in the same or varyingdegree at several sites in a given polypeptide. Also, a givenpolypeptide may contain many types of modifications. In general, as usedherein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

VARIANT(S) of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail. (1) A polynucleotide that differs in nucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the nucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical. As noted below,changes in the nucleotide sequence of the variant may be silent. Thatis, they may not alter the amino acids encoded by the polynucleotide.Where alterations are limited to silent changes of this type a variantwill encode a polypeptide with the same amino acid sequence as thereference. Also as noted below, changes in the nucleotide sequence ofthe variant may alter the amino acid sequence of a polypeptide encodedby the reference polynucleotide. Such nucleotide changes may result inamino acid substitutions, additions, deletions, fusions and truncationsin the polypeptide encoded by the reference sequence, as discussedbelow. (2) A polypeptide that differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference and the variant are closely similaroverall and, in many region, identical. A variant and referencepolypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions, fusions and truncations, which maybe present in any combination. An “N” in a polynucleotide sequence ofthe invention means polynucleotides comprising A, C, G or T at thisposition are included as embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel spo-rel polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel spo-rel gene of Staphylococcus aureus, whichis related by amino acid sequence homology to Streptococcus equisimilisrel protein polypeptide. The invention relates especially to spo-relhaving the nucleotide and amino acid sequences set out in FIG. 1 [SEQ IDNO:1] and FIG. 2 [SEQ ID NO:2] respectively, and to the spo-relnucleotide and amino acid sequences of the DNA in NCIMB Deposit No.40771, which is herein referred to as “the deposited bacterial strain”or as the “DNA of the deposited bacterial strain.” It will beappreciated that the nucleotide and amino acid sequences set out inFIGS. 1 [SEQ ID NO:1] and 2 [SEQ ID NO:2] were obtained by sequencingthe DNA of the deposited bacterial strain. Hence, the sequence ofspo-rel of the deposited bacterial strain is controlling as to anydiscrepancies between it (and the sequence it encodes) and the sequencesof FIG. 1 [SEQ ID NO: 1] and FIG. 2 [SEQ ID NO:2].

The invention also provides spo-rel having the nucleotide and amino acidsequences set out in FIG. 3 [SEQ ID NO:7] and FIG. 4 [SEQ ID NO:8]respectively

Techniques are available to evaluate temporal gene expression inbacteria, particularly as it applies to viability under laboratory andhost infection conditions. A number of methods can be used to identifygenes which are essential to survival per se, or essential to theestablishing/maintenance of an infection. Identification of expressionof a sequence by one of these methods yields additional informationabout its function and permits the selection of such sequence forfurther development as a screening target. Briefly, these approachesinclude:

1) Signature Tagged Mutagenesis (STM)

This technique is described by Hensel et al., Science 269:400-403(1995), the contents of which is incorporated by reference forbackground purposes. Signature tagged mutagenesis identifies genesnecessary for the establishment/maintenance of infection in a giveninfection model.

The basis of the technique is the random mutagenesis of target organismby various means (e.g., transposons) such that unique DNA sequence tagsare inserted in close proximity to the site of mutation. The tags from amixed population of bacterial mutants and bacteria recovered from aninfected hosts are detected by amplification, radiolabeling andhybridization analysis. Mutants attenuated in virulence are revealed byabsence of the tag from the pool of bacteria recovered from infectedhosts.

In Staphylococcus aureus, because the transposon system is less welldeveloped, a more efficient way of creating the tagged mutants is to usethe insertion-duplication mutagenesis technique as described by Morrisonet al, J. Bacteriol. 159:870 (1984) the contents of which isincorporated by reference for background purposes.

2) In Vivo Expression Technology (IVET)

This technique is described by Camilli et al., Proc. Nat'l. Acad. Sci.USA. 91:2634-2638 (1994), the contents of which is incorporated byreference for background purposes. IVET identifies genes up-regulatedduring infection when compared to laboratory cultivation, implying animportant role in infection. Sequences identified by this technique areimplied to have a significant role in infectionestablishment/maintenance.

In this technique random chromosomal fragments of target organism arecloned upstream of a promoter-less reporter gene in a plasmid vector.The pool is introduced into a host and at various times after infectionbacteria may be recovered and assessed for the presence of reporter geneexpression. The chromosomal fragment carried upstream of an expressedreporter gene should carry a promoter or portion of a gene normallyupregulated during infection. Sequencing upstream of the reporter geneallows identification of the up regulated gene.

3) Differential Display

This technique is described by Chuang et al, J. Bacteriol 175:2026-2036(1993), the contents of which is incorporated by reference forbackground purposes. This method identifies those genes which areexpressed in an organism by identifying mRNA present usingrandomly-primed RT-PCR. By comparing pre-infection and post infectionprofiles, genes up and down regulated during infection can be identifiedand the RT-PCR product sequenced and matched to library sequences.

4) Generation of conditional lethal mutants by transposon mutagenesis.

This technique, described by de Lorenzo, V. et al, Gene 123:17-24(1993); Neuwald, A. F. et al., Gene 125: 69-73(1993); and Takiff, H. E.et al., J. Bacteriol. 174:1544-1553(1992), the contents of which isincorporated by reference for background purposes, identifies geneswhose expression are essential for cell viability.

In this technique transposons carrying controllable promoters, whichprovide transcription outward from the transposon in one or bothdirections, are generated. Random insertion of these transposons intotarget organisms and subsequent isolation of insertion mutants in thepresence of inducer of promoter activity ensures that insertions whichseparate promoter from coding region of a gene whose expression isessential for cell viability will be recovered. Subsequent replicaplating in the absence of inducer identifies such insertions, since theyfail to survive. Sequencing of the flanking regions of the transposonallows identification of site of insertion and identification of thegene disrupted. Close monitoring of the changes in cellularprocesses/morphology during growth in the absence of inducer yieldsinformation on likely function of the gene. Such monitoring couldinclude flow cytometry (cell division, lysis, redox potential, DNAreplication), incorporation of radiochemically labeled precursors intoDNA, RNA, protein, lipid, peptidoglycan, monitoring reporter enzyme genefusions which respond to known cellular stresses.

5) Generation of conditional lethal mutants by chemical mutagenesis.

This technique is described by Beckwith, J., Methods in Enzymology 204:3-18(1991), the contents of which are incorporated herein by referencefor background purposes. In this technique random chemical mutagenesisof target organism, growth at temperature other than physiologicaltemperature (permissive temperature) and subsequent replica plating andgrowth at different temperature (e.g. 42° C. to identify ts, 25° C. toidentify cs) are used to identify those isolates which now fail to grow(conditional mutants). As above close monitoring of the changes upongrowth at the non-permissive temperature yields information on thefunction of the mutated gene. Complementation of conditional lethalmutation by library from target organism and sequencing of complementinggene allows matching with library sequences.

Each of these techniques may have advantages or disadvantage dependingon the particular application. The skilled artisan would choose theapproach that is the most relevant with the particular end use in mind.For example, some genes might be recognised as essential for infectionbut in reality are only necessary for the initiation of infection and sotheir products would represent relatively unattractive targets forantibacterials developed to cure established and chronic infections.

6) RT-PCR

Bacterial messenger RNA, preferably that of Staphylococcus aureus, isisolated from bacterial infected tissue e.g. 48 hour murine lunginfections, and the amount of each mRNA species assessed by reversetranscription of the RNA sample primed with random hexanucleotidesfollowed by PCR with gene specific primer pairs. The determination ofthe presence and amount of a particular mRNA species by quantificationof the resultant PCR product provides information on the bacterial geneswhich are transcribed in the infected tissue. Analysis of genetranscription can be carried out at different times of infection to gaina detailed knowledge of gene regulation in bacterial pathogenesisallowing for a clearer understanding of which gene products representtargets for screens for novel antibacterials. Because of the genespecific nature of the PCR primers employed it should be understood thatthe bacterial mRNA preparation need not be free of mammalian RNA. Thisallows the investigator to carry out a simple and quick RNA preparationfrom infected tissue to obtain bacterial mRNA species which are veryshort lived in the bacterium (in the order of 2 minute halflives).Optimally the bacterial mRNA is prepared from infected murine lungtissue by mechanical disruption in the presence of TRIzole (GIBCO-BRL)for very short periods of time, subsequent processing according to themanufacturers of TRIzole reagent and DNAase treatment to removecontaminating DNA. Preferably the process is optimized by finding thoseconditions which give a maximum amount of bacterial 16S ribosomal RNA,preferably that of Staphylococcus aureus, as detected by probingNortherns with a suitably labeled sequence specific oligonucleotideprobe. Typically a 5′, dye labelled primer is used in each PCR primerpair in a PCR reaction which is terminated optimally between 8 and 25cycles. The PCR products are separated on 6% polyacrylamide gels withdetection and quantification using GeneScanner (manufactured by ABI).Using RT-PCR it was determined that the polynucleotide set forth in SEQID NO:1 is expressed in vivo.

Use of the of these technologies when applied to the sequences of thepresent invention enables identification of bacterial protein speciesexpressed during infection, inhibitors of which would have utility inanti-bacterial therapy.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the spo-rel polypeptidehaving the deduced amino acid sequence of FIG. 2 [SEQ ID NO:2].

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 [SEQ ID NO:1], a polynucleotide of thepresent invention encoding spo-rel polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloning andsequencing genomic DNA fragments from Staphylococcus aureus WCUH 29cells as starting material, followed by obtaining a full length clone.For example, to obtain a polynucleotide of the invention sequence, suchas that sequence given in FIG. 1 [SEQ ID NO: 1] typically a library ofclones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E. coli orsome other suitable host is probed with a radiolabeled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing high stringency washes. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York(1989). (see Screening By Hybridization 1.90 and Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Mustrative of the invention, thepolynucleotide set out in FIG. 1 [SEQ ID NO:1] was discovered in a DNAlibrary derived from Staphylococcus aureus WCUH 29.

Spo-rel of the invention is structurally related to other proteins ofthe relA/spoT gene family, as shown by the results of sequencing the DNAencoding spo-rel of the deposited bacterial strain. The DNA sequencethus obtained is set out in FIG. 1 [SEQ ID NO:1]. It contains an openreading frame encoding a protein having about the number of amino acidresidues set forth in FIG. 2 [SEQ ID NO:2] with a deduced molecularweight that can be calculated using amino acid residue molecular weightvalues well known in the art. The protein exhibits greatest homology toStreptococcus equisimilis rel protein protein among known proteins.spo-rel of FIG. 2 [SEQ ID NO:2] has about 50% identity over its entirelength and about 69% similarity over its entire length with the aminoacid sequence of Streptococcus equisimilis rel protein.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1 [SEQ ID NO:1].It also may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptide of FIG. 2 [SEQ ID NO:2].

Polynucleotides of the present invention which encode the polypeptide ofFIG. 2 [SEQ ID NO:2] may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals, for example), ribosome binding, mRNA stabilityelements, and additional coding sequence which encode additional aminoacids, such as those which provide additional functionalities. Thus, forinstance, the polypeptide may be fused to a marker sequence, such as apeptide, which facilitates purification of the fused polypeptide. Incertain embodiments of this aspect of the invention, the marker sequenceis a hexa-histidine peptide, such as the tag provided in the pQE vector(Qiagen, Inc.), among others, many of which are commercially available.As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), for instance, hexa-histidine provides for convenientpurification of the fusion protein. The HA tag may also be used tocreate fusion proteins and corresponds to an epitope derived ofinfluenza hemagglutinin protein, which has been described by Wilson etal, Cell 37: 767 (1984), for instance. Polynucleotides of the inventionalso include, but are not limited to, polynucleotides comprising astructural gene and its naturally associated genetic elements.

In accordance with the foregoing, the term “polynucleotide encoding apolypeptide” as used herein encompasses polynucleotides which include asequence encoding a polypeptide of the present invention, particularlybacterial, and more particularly the Staphylococcus aureus spo-relhaving the amino acid sequence set out in FIG. 2 [SEQ ID NO:2]. The termencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 2 [SEQ ID NO:2]. A variant of the polynucleotide may be a naturallyoccurring variant such as a naturally occurring allelic variant, or itmay be a variant that is not known to occur naturally. Suchnon-naturally occurring variants of the polynucleotide may be made bymutagenesis techniques, including those applied to polynucleotides,cells or organisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of spo-rel set out in FIG. 2 [SEQ ID NO:2]; variants, analogs,derivatives and fragments thereof, and fragments of the variants,analogs and derivatives.

Further particularly preferred in this regard are polynucleotidesencoding spo-rel variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of spo-rel polypeptide of FIG. 2 [SEQ ID NO:2] in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, deleted or added, in any combination. Especiallypreferred among these are silent substitutions, additions and deletions,which do not alter the properties and activities of spo-rel. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polynucleotides encoding polypeptides having theamino acid sequence of FIG. 2 [SEQ ID NO:2], without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare at least 70% identical over their entire length to a polynucleotideencoding spo-rel polypeptide having the amino acid sequence set out inFIG. 2 [SEQ ID NO:2], and polynucleotides which are complementary tosuch polynucleotides. Alternatively, most highly preferred arepolynucleotides that comprise a region that is at least 80% identicalover their entire length to a polynucleotide encoding spo-relpolypeptide of the Staphylococcus aureus DNA of the deposited bacterialstrain and polynucleotides complementary thereto. In this regard,polynucleotides at least 90% identical over their entire length to thesame are particularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

The present invention further relates to polynucleotides which hybridizeto the hereinabove-described sequences if there is at least 50% andpreferably at least 70% identity between the sequences, particularlypolynucleotides which hybridize to the polynucleotides set forth in FIG.3 [SEQ ID NO:7] and/or FIG. 1 [SEQ ID NO:1]. The present inventionparticularly relates to Staphylococcal polynucleotides which hybridizeunder stringent conditions to the hereinabove-described polynucleotides,particularly polynucleotides which hybridize to the polynucleotide setforth in FIG. 3 [SEQ ID NO:7] and/or FIG. 1 [SEQ ID NO:1]. As hereinused, the term “stringent conditions” means hybridization will occuronly if there is at least 95% and preferably at least 97% identitybetween the sequences.

Preferred embodiments in this respect, moreover, are polynucleotideswhich encode polypeptides which retain substantially the same biologicalfunction or activity as the mature polypeptide encoded by the DNA ofFIG. 1 [SEQ ID NO:1], such as, for example, the polypeptide encoded bynucleic acids 700 to 2907 of FIG. 1 [SEQ ID NO: 1].

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as a hybridization probe for RNA, cDNA and genomicDNA to isolate full-length cDNAs and genomic clones encoding spo-rel andto isolate cDNA and genomic clones of other genes that have a highsequence similarity to the spo-rel gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

For example, the coding region of the spo-rel gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofcDNA, genomic DNA or mRNA to determine which members of the library theprobe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsof and diagnostics for disease, particularly human disease, as furtherdiscussed herein relating to polynucleotide assays, inter alia.

The polynucleotides of the invention that are oligonucleotides,including SEQ ID NOS:3 and 4, derived from the sequences of SEQ ID NOS:1 and 2 may be used in the processes herein as described, but preferablyfor PCR, to determine whether or not the Staphylococcus aureus genesidentified herein in whole or in part are transcribed in infectedtissue. It is recognized that such sequences will also have utility indiagnosis of the stage of infection and type of infection the pathogenhas attained.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may allowprotein transport, may lengthen or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited Materials

The deposit has been made under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Micro-organisms for Purposesof Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

A deposit containing a Staphylococcus aureus bacterial strain (WCUH29)has been deposited with the National Collections of Industrial andMarine Bacteria Ltd. (NCIMB), 23 St. Machar Drive, Aberdeen AB2 IRY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771. TheStaphylococcus aureus bacterial strain of the deposit is referred toherein as “the deposited bacterial strain” or as “the DNA of thedeposited bacterial strain” and is referred to as “NCIMB 40771” upondeposit.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to a spo-rel polypeptide which hasa deduced amino acid sequence of 735 amino acids in length, as set forthin FIG. 2 [SEQ ID NO:2], and has a deduced molecular weight of 84,536.52Daltons.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms “fragment,” “derivative” and “analog” whenreferring to the polypeptide of FIG. 2 [SEQ ID NO:2], means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 2 [SEQ IDNO:2] may be (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of spo-rel setout in FIG. 2 [SEQ ID NO:2], variants, analogs, derivatives andfragments thereof, and variants, analogs and derivatives of thefragments. Alternatively, particularly preferred embodiments of theinvention in this regard are polypeptides having the amino acid sequenceof the spo-rel, variants, analogs, derivatives and fragments thereof,and variants, analogs and derivatives of the fragments.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the spo-rel polypeptide ofFIG. 2 [SEQ ID NO:2], in which several, a few, 5 to 10, 1 to 5, 1 to 3,2, 1 or no amino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the spo-rel. Also especially preferred in this regard areconservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 2 [SEQ ID NO:2] withoutsubstitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide ofFIG. 2 [SEQ ID NO:2] (in particular the mature polypeptide) as well aspolypeptides which have at least 70% identity to the polypeptide of FIG.2 [SEQ ID NO:2], preferably at least 80% identity to the polypeptide ofFIG. 2 [SEQ ID NO:2], and more preferably at least 90% similarity (morepreferably at least 90% identity) to the polypeptide of FIG. 2 [SEQ IDNO:2] and still more preferably at least 95% similarity (still morepreferably at least 95% identity) to the polypeptide of FIG. 2 [SEQ IDNO:2] and also include portions of such polypeptides with such portionof the polypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

The polypeptides of the present invention include the polypeptide ofFIG. 4 [SEQ ID NO:8] as well as polypeptides which have at least 70%identity to the polypeptide of FIG. 4 [SEQ ID NO:8], preferably at least80% identity to the polypeptide of FIG. 4 [SEQ ID NO:8], and morepreferably at least 90% similarity (more preferably at least 90%identity) to the polypeptide of FIG. 4 [SEQ ID NO:8] and still morepreferably at least 95% similarity (still more preferably at least 95%identity) to the polypeptide of FIG. 4 [SEQ ID NO:8] and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.

Fragments

Also among preferred embodiments of this aspect of the present inventionare polypeptides comprising fragments of spo-rel, most particularlyfragments of spo-rel having the amino acid set out in FIG. 2 [SEQ IDNO:2], and fragments of variants and derivatives of the spo-rel of FIG.2 [SEQ ID NO:2].

In this regard a fragment is a polypeptide having an amino acid sequencethat entirely is the same as part but not all of the amino acid sequenceof the aforementioned spo-rel polypeptides and variants or derivativesthereof.

Such fragments may be “free-standing,” i.e., not part of or fused toother amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a spo-relpolypeptide of the present comprised within a precursor polypeptidedesigned for expression in a host and having heterologous pre andpro-polypeptide regions fused to the amino terminus of the spo-relfragment and an additional region fused to the carboxyl terminus of thefragment. Therefore, fragments in one aspect of the meaning intendedherein, refers to the portion or portions of a fusion polypeptide orfusion protein derived from spo-rel.

Representative examples of polypeptide fragments of the invention,include, for example, fragments from about amino acid number 1-20,21-40, 41-60, 61-80, 81-100, and in 20 amino acid increments from101-735, and any combination of any of these 20 amino acid fragments. Inthis context “about” herein includes the particularly recited rangeslarger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid ateither extreme or at both extremes.

Preferred fragments of the invention include, for example, truncationpolypeptides of spo-rel. Truncation polypeptides include spo-relpolypeptides having the amino acid sequence of FIG. 2 [SEQ ID NO:2], orof variants or derivatives thereof, except for deletion of a continuousseries of residues (that is, a continuous region, part or portion) thatincludes the amino terminus, or a continuous series of residues thatincludes the carboxyl terminus or, as in double truncation mutants,deletion of two continuous series of residues, one including the aminoterminus and one including the carboxyl terminus. Fragments having thesize ranges set out about also are preferred embodiments of truncationfragments, which are especially preferred among fragments generally.Degradation forms of the polypeptides of the invention in a host cell,particularly a Staphylococcus, are also preferred.

Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of spo-rel.Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions of spo-rel, and combinations of such fragments.

Preferred regions are those that mediate activities of spo-rel. Mosthighly preferred in this regard are fragments that have a chemical,biological or other activity of spo-rel, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Further preferred polypeptide fragments are those that areantigenic or immunogenic in an animal, especially in a human.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespond to the preferred fragments, as discussed above.

Vectors, Host Cells, Expression

The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. Introduction of apolynucleotides into the host cell can be affected by calcium phosphatetransfection, DEAE-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

Polynucleotide constructs in host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, asingle or double-stranded RNA or DNA viral vector. Plasmids generallyare designated herein by a lower case p preceded and/or followed bycapital letters and/or numbers, in accordance with standard namingconventions that are familiar to those of skill in the art. Startingplasmids disclosed herein are either commercially available, publiclyavailable, or can be constructed from available plasmids by routineapplication of well known, published procedures. Many plasmids and othercloning and expression vectors that can be used in accordance with thepresent invention are well known and readily available to those of skillin the art.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include, among others,chromosomal, episomal and virus-derived vectors, e.g., vectors derivedfrom bacterial plasmids, from bacteriophage, from transposons, fromyeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids, all may be used for expression inaccordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques, such as, for example,those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York (1989).

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct mRNA transcription. Representatives of such promotersinclude, but are not limited to, the phage lambda PL promoter, the E.coli lac, trp and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription, such as transcription factors, repressor binding sitesand termination, among others.

Vectors for propagation and expression generally will include selectablemarkers and amplification regions, such as, for example, those set forthin Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York(1989).

Representative examples of appropriate hosts include bacterial cells,such as streptococci, staphylococci, E. coli, streptomyces and Bacillussubtilis cells; fungal cells, such as yeast cells and Aspergillus cells;insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animalcells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanomacells; and plant cells.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia, and pBR322 (ATCC 37017). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSK3 pBPV, pMSG and pSVL available from PharmaciaThese vectors are listed solely by way of illustration of the manycommercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase (“CAT”) transcription unit,downstream of restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the cat gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable, such as pKK232-8 and pCM7. Promoters for expression ofpolynucleotides of the present invention include not only well known andreadily available promoters, but also promoters that readily may beobtained by the foregoing technique, using a reporter gene.

Among known prokaryotic promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous sarcoma virus (“RSV”), and metallothionein promoters, suchas the mouse metallothionein-I promoter.

Recombinant expression vectors will include, for example, origins ofreplication, a promoter preferably derived from a highly-expressed geneto direct transcription of a downstream structural sequence, and aselectable marker to permit isolation of vector containing cells afterexposure to the vector.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5′ to aribosome binding site. The ribosome binding site will be 5′ to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiation codon. Also, generally, there will be a translationstop codon at the end of the polypeptide and there will be apolyadenylation signal in constructs for use in eukaryotic hosts.Transcription termination signal appropriately disposed at the 3′ end ofthe transcribed region may also be included in the polynucleotideconstruct.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N- or C-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunolglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another proteinor part thereof. In drug discovery, for example, proteins have beenfused with antibody Fc portions for the purpose of high-throughputscreening assays to identify antagonists. See, D. Bennett et al.,Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson etal, The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471(1995).

Cells typically then are harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents, such methods arewell know to those skilled in the art.

Mammalian expression vectors may comprise an origin of replication, asuitable promoter and enhancer, and also any necessary ribosome bindingsites, polyadenylation regions, splice donor and acceptor sites,transcriptional termination sequences, and 5′ flanking non-transcribedsequences that are necessary for expression.

spo-rel polypeptide can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatographyis employed for purification. Well known techniques for refoldingprotein may be employed to regenerate active conformation when thepolypeptide is denatured during isolation and or purification.

Polynucleotide Assays

This invention is also related to the use of the spo-rel polynucleotidesto detect complementary polynucleotides such as, for example, as adiagnostic reagent. Detection of spo-rel in a eukaryote, particularly amammal, and especially a human, will provide a diagnostic method fordiagnosis of a disease. Eukaryotes (herein also “individual(s)”),particularly mammals, and especially humans, infected with an organismcomprising the spo-rel gene may be detected at the DNA level by avariety of techniques. Nucleic acids for diagnosis may be obtained froman infected individual's cells and tissues, such as bone, blood, muscle,cartilage, and skin. Genomic DNA may be used directly for detection ormay be amplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166 (1986) prior to analysis. RNA or cDNA may also be used in thesame ways. As an example, PCR primers complementary to the nucleic acidencoding spo-rel can be used to identify and analyze spo-rel presenceand/or expression. Using PCR, characterization of the strain ofprokaryote present in a eukaryote, particularly a mammal, and especiallya human, may be made by an analysis of the genotype of the prokaryotegene. For example, deletions and insertions can be detected by a changein size of the amplified product in comparison to the genotype of areference sequence. Point mutations can be identified by hybridizingamplified DNA to radiolabeled spo-rel RNA or alternatively, radiolabeledspo-rel antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsalso may be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or another amplification method. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic characterization based on DNA sequence differences may beachieved by detection of alteration in electrophoretic mobility of DNAfragments in gels, with or without denaturing agents. Small sequencedeletions and insertions can be visualized by high resolution gelelectrophoresis. DNA fragments of different sequences may bedistinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science , 230: 1242(1985)).

Sequence changes at specific locations also may be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., Proc. Natl, Acad. Sci., USA, 85:4397-4401(1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations also can be detected by in situ analysis.

Cells carrying mutations or polymorphisms in the gene of the presentinvention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to the nucleicacid encoding spo-rel can be used to identify and analyze mutations.Examples of representative primers are shown below in Table

TABLE 1 Primers used for amplification of spo-rel polynucleotides SEQ IDNO PRIMER SEOUENCE 3 5′-CCGCCATGGGTAACAACGAATATCCATATAGT-3′ 45′-CGGGATCCGCACCTCTAGTTCCAAACTCT-3′ 5 5′-agatacgtac tgaaatgg-3′ 65′-cctgtgattc gcataagc-3′

These primers may be used for amplifying spo-rel DNA isolated from asample derived from an individual. The invention also provides theprimers of Table 1 with 1, 2, 3 or 4 nucleotides removed from the 5′and/or the 3′ end. The primers may be used to amplify the gene isolatedfrom the individual such that the gene may then be subject to varioustechniques for elucidation of the DNA sequence. In this way, mutationsin the DNA sequence may be diagnosed.

The invention provides a process for diagnosing, disease, preferablybacterial infections, more preferably Staphylococcus aureus, and mostpreferably upper respiratory tract (e.g. otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.empyema, lung abscess), cardiac (e.g. infective endocarditis),gastrointestinal (e.g. secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g.blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis), and bone and joint (e.g. septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of FIG. 1 [SEQ ID NO: 1]. Increased expression ofspo-rel polynucleotide can be measured using any on of the methods wellknown in the art for the quantation of polynucleotides, such as, forexample, PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods.

Polypeptide Assays

The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of spo-relprotein in cells and tissues, including determination of normal andabnormal levels. Thus, for instance, a diagnostic assay in accordancewith the invention for detecting over-expression of spo-rel proteincompared to normal control tissue samples may be used to detect thepresence of an infection, for example. Assay techniques that can be usedto determine levels of a spo-rel protein, in a sample derived from ahost are well-known to those of skill in the art. Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays. Among these ELISAs frequently are preferred.An ELISA assay initially comprises preparing an antibody specific tospo-rel, preferably a monoclonal antibody. In addition a reporterantibody generally is prepared which binds to the monoclonal antibody.The reporter antibody is attached a detectable reagent such asradioactive, fluorescent or enzymatic reagent, in this examplehorseradish peroxidase enzyme.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can be used as an immunogen to produceantibodies thereto. The present invention includes, for examplesmonoclonal and polyclonal antibodies, chimeric, single chain, andhumanized antibodies, as well as Fab fragments, or the product of an Fabexpression library.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique known in the artwhich provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies toimmunogenic polypeptide products of this invention. Also, transgenicmice, or other organisms such as other mammals, may be used to expresshumanized antibodies to immunogenic polypeptide products of thisinvention.

Alternatively phage display technology could be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-Fbp or from naive libraries (McCafferty, J.et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992)Biotechnology 10, 779-783). The affinity of these antibodies can also beimproved by chain shuffling (Clackson, T. et al., (1991) Nature 352,624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope-termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Thus, among others, antibodies against spo-rel may be employed toinhibit and/or treat infections, particularly bacterial infections andespecially upper respiratory tract (e.g. otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.empyema, lung abscess),cardiac (e.g. infective endocarditis),gastrointestinal (e.g. secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g.blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal andorbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g. impetigo, folliculitis , cutaneous abscesses, cellulitis,wound infection, bacterial myositis), and bone and joint (e.g. septicarthritis, osteomyelitis).

Polypeptide derivatives include antigenically, epitopically orimmunologically equivalent derivatives which form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognised by certain antibodies which, when raised to theprotein or polypeptide according to the present invention, interferewith the immediate physical interaction between pathogen and mammalianhost. The term “immunologically equivalent derivative” as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between pathogen andmammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation , with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably the antibody or derivative thereof is modified to make itless immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized”; where thecomplimentarily determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody , for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem1989:264,16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS, 1986:83,9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS 1984:81,5849).

spo-rel-binding Molecules and Assays

This invention also provides a method for identification of molecules,such as binding molecules, that bind spo-rel. Genes encoding proteinsthat bind spo-rel, can be identified by numerous methods known to thoseof skill in the art, for example, ligand panning and FACS sorting. Suchmethods are described in many laboratory manuals such as, for instance,Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).Also, a labeled ligand can be photoaffinity linked to a cell extract.

Polypeptides of the invention also can be used to assess spo-rel bindingcapacity of spo-rel-binding molecules, in cells or in cell-freepreparations.

Polypeptides of the invention may also be used to assess the binding orsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics.

Antagonists and Agonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofspo-rel polypeptides or polynucleotides, such as its interaction withspo-rel-binding molecules.

For example, to screen for agonists or antagonists, a synthetic reactionmix, a cellular compartment, such as a membrane, cell envelope or cellwall, or a preparation of any thereof, may be prepared from a cell thatexpresses a molecule that binds spo-rel. The preparation is incubatedwith labeled spo-rel in the absence or the presence of a candidatemolecule which may be a spo-rel agonist or antagonist. The ability ofthe candidate molecule to bind the binding molecule is reflected indecreased binding of the labeled ligand. Molecules which bindgratuitously, i.e., without inducing the effects of spo-rel on bindingthe spo-rel binding molecule, are most likely to be good antagonists.Molecules that bind well and elicit effects that are the same as orclosely related to spo-rel are agonists.

spo-rel-like effects of potential agonists and antagonists may bymeasured, for instance, by determining activity of a reporter systemfollowing interaction of the candidate molecule with a cell orappropriate cell preparation, and comparing the effect with that ofspo-rel or molecules that elicit the same effects as spo-rel. Reportersystems that may be useful in this regard include but are not limited tocolorimetric labeled substrate converted into product, a reporter genethat is responsive to changes in spo-rel activity, and binding assaysknown in the art.

Another example of an assay for spo-rel antagonists is a competitiveassay that combines spo-rel and a potential antagonist withmembrane-bound spo-rel-binding molecules, recombinant spo-rel bindingmolecules, natural substrates or ligands, or substrate or ligandmimetics, under appropriate conditions for a competitive inhibitionassay. spo-rel can be labeled, such as by radioactivity or acolorimetric compound, such that the number of spo-rel molecules boundto a binding molecule or converted to product can be determinedaccurately to assess the effectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducingspo-rel-induced activities, thereby preventing the action of spo-rel byexcluding spo-rel from binding.

Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules.

Other potential antagonists include antisense molecules (see Okano, J.Neurochem 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Other potential antagonists alsoinclude compounds related to and derivatives of spo-rel.

In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the initial physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used: i) in the prevention of adhesion ofbacteria, in particular gram positive bacteria, to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; ii) to block guanosine 3′,5′-bis(diphosphate) synthetase/hydrolase protein mediated mammalian cellinvasion by, for example, initiating phosphorylation of mammaliantyrosine kinases (Rosenshine et al, Infect, Immun. 60:2211 (1992); iii)to block bacterial adhesion between mammalian extracellular matrixproteins and bacterial guanosine 3′, 5′-bis(diphosphate)synthetase/hydrolase proteins which mediate tissue damage; iv) to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein uponexpression can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The antagonists and agonists may be employed for instance to inhibitupper respiratory tract (e.g. otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g. empyema, lungabscess),cardiac (e.g. infective endocarditis), gastrointestinal (e.g.secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS(e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal & orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g. epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis), and bone and joint (e.g. septicarthritis, osteomyelitis)

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with spo-rel, or a fragment orvariant thereof, adequate to produce antibody to protect said individualfrom infection, particularly bacterial infection and most particularlyStaphylococcus infections. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises, through gene therapy, delivering gene encoding spo-rel, or afragment or a variant thereof, for expressing spo-rel, or a fragment ora variant thereof in vivo in order to induce an immunological responseto produce antibody to protect said individual from disease.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable or having inducedwithin it an immunological response, induces an immunological responsein such host to a spo-rel or protein coded therefrom, wherein thecomposition comprises a recombinant spo-rel or protein coded therefromcomprising DNA which codes for and expresses an antigen of said spo-relor protein coded therefrom.

The spo-rel or a fragment thereof may be fused with co-protein which maynot by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. Thus fused recombinant protein, preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of Staphylococcus aureus infection inmammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused e.g. by mechanical, chemical or thermal damage or by implantationof indwelling devices, or wounds in the mucous membranes, such as themouth, mammary glands, urethra or vagina.

The present invention also includes a vaccine formulation whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certainspo-rel, it is to be understood that this covers fragments of thenaturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

Compositions

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or the agonists or antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

Kits

The invention further relates to diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, dose is from about 10 μg/kg to about 1mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

In-dwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

The composition of the invention may be administered by injection toachieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent Staphylococcus wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 μg/ml to 10mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response.

A suitable unit dose for vaccination is 0.5-5 μg/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks.

With the indicated dose range, no adverse toxicological effects will beobserved with the compounds of the invention which would preclude theiradministration to suitable individuals.

The antibodies described above may also be used as diagnostic reagentsto detect the presence of bacteria containing the guanosine 3′,5′-bis(diphosphate) synthetase/hydrolase protein.

In order to facilitate understanding of the following example certainfrequently occurring methods and/or terms will be described.

EXAMPLES

The present invention is further described by the following examples.These exemplification's, while illustrating certain specific aspects ofthe invention, do not portray the limitations or circumscribe the scopeof the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples were carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

Example 1 Library Production

The polynucleotide having the DNA sequence given in SEQ ID NO: 1 wasobtained from a library of clones of chromosomal DNA of Staphylococcusaureus in E. coli. In some cases the sequencing data from two or moreclones containing overlapping Staphylococcus aureus DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO:1. Libraries may beprepared by routine methods, for example: Methods 1 and 2 below.

Total cellular DNA is isolated from Staphylococcus aureus WCUH 29according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

Example 2 Complementation of an E. coli ppGpp-less Strain

An E. coli mutant lacking both ppGpp synthetases, encoded by the relAand spoT genes, cannot grow in the absence of amino acid supplementation(Xiao et al., 1991). Any expressed gene that encodes a ppGpp synthetaseis expected to provide sufficient ppGpp to allow growth on media lackingamino acids (Gentry and Cashel, 1996; Mechold et al. 1996). AStaphylococcus areus spo-rel gene is cloned into pBluescript andtransformed into an E. coli Dre/A DspoT strain (Xiao et al., 1991).Resulting transformants are screened for their ability to grow on M63medium supplemented with glucose (0.2%). Complementation is scored after16 h growth at 37° C.

Example 2 References

Gentry, D. R., and M. Cashel. 1996. Mutational analysis of theEscherichia coli spoT gene identifies distinct but overlapping regionsinvolved in ppGpp synthesis and degradation. Mol. Microbiol.19:1373-1384.

Mechold, U., M. Cashel, K. Steiner, D. Gentry, and H. Malke. 1996.Functional analysis of a relA/spoT gene homolog of Streptococcusequisimilis. J. Bacteriol. 178:1401-1411.

Xiao, H., M. Kalman, K. Ikehara, S. Zemel, G. Glaser, and M. Cashel.1991. Residual guanosine 3′, 5′-bispyrophosphate synthetic activity of arelA null mutants can be eliminated by spoT null mutations. J. Biol.Chem. 266: 5980-5990.

Example 3 The Determination of Expression During Infection of a Genefrom Staphylococcus aureus

Necrotic fatty tissue from a four day groin infection of Staphylococcusaureus WCUH29 in the mouse is efficiently disrupted and processed in thepresence of chaotropic agents and RNAase inhibitor to provide a mixtureof animal and bacterial RNA. The optimal conditions for disruption andprocessing to give stable preparations and high yields of bacterial RNAare followed by the use of hybridisation to a radiolabelledoligonucleotide specific to Staphylococcus aureus 16S RNA on Northernblots. The RNAase free, DNAase free, DNA and protein free preparationsof RNA obtained are suitable for Reverse Transcription PCR (RT-PCR)using unique primer pairs designed from the sequence of each gene ofStaphylococcus aureus WCUH29.

a) Isolation of Tissue Infected with Staphylococcus aureus WCUH29 from aMouse Animal Model of Infection

10 ml. volumes of sterile nutrient broth (No.2 Oxoid) are seeded withisolated, individual colonies of Staphylococcus aureus WCUH29 from anagar culture plate. The cultures are incubated aerobically (staticculture) at 37° C. for 16-20 hours . 4 week old mice (female, 18 g-22 g,strain MF1) are each infected by subcutaneous injection of 0.5 ml. ofthis broth culture of Staphylococcus aureus WCUH29 (diluted in broth toapproximately 10⁸ cfu/ml.) into the anterior, right lower quadrant(groin area). Mice should be monitored regularly during the first 24hours after infection, then daily until termination of study. Animalswith signs of systemic infection, i.e. lethargy, ruffled appearance,isolation from group, should be monitored closely and if signs progressto moribundancy, the animal should be culled immediately.

Visible external signs of lesion development will be seen 24-48 h afterinfection. Examination of the abdomen of the animal will show the raisedoutline of the abscess beneath the skin. The localised lesion shouldremain in the right lower quadrant, but may occasionally spread to theleft lower quadrant, and superiorly to the thorax. On occasions, theabscess may rupture through the overlying skin layers. In such cases theaffected animal should be culled immediately and the tissues sampled ifpossible. Failure to cull the animal may result in the necrotic skintissue overlying the abscess being sloughed off, exposing the abdominalmuscle wall.

Approximately 96 hours after infection, animals are killed using carbondioxide asphyxiation. To minimise delay between death and tissueprocessing /storage, mice should be killed individually rather than ingroups. The dead animal is placed onto its back and the fur swabbedliberally with 70% alcohol. An initial incision using scissors is madethrough the skin of the abdominal left lower quadrant, travellingsuperiorly up to, then across the thorax. The incision is completed bycutting inferiorly to the abdominal lower right quadrant. Care should betaken not to penetrate the abdominal wall. Holding the skin flap withforceps, the skin is gently pulled way from the abdomen. The exposedabscess, which covers the peritoneal wall but generally does notpenetrate the muscle sheet completely, is excised, taking care not topuncture the viscera

The abscess/muscle sheet and other infected tissue may require cuttingin sections, prior to flash-freezing in liquid nitrogen, therebyallowing easier storage in plastic collecting vials.

b) Isolation of Staphylococcus Aureus WCUH29 RNA from Infected TissueSamples

4-6 infected tissue samples(each approx 0.5-0.7g) in 2 ml screw-captubes are removed from −80° C. storage into a dry ice ethanol bath In amicrobiological safety cabinet the samples are disrupted individuallywhilst the remaining samples are kept cold in the dry ice ethanol bath.To disrupt the bacteria within the tissue sample 1 ml of TRIzol Reagent(Gibco BRL, Life Technologies) is added followed by enough 0.1 mmzirconia/silica beads to almost fill the tube, the lid is replacedtaking care not to get any beads into the screw thread so as to ensure agood seal and eliminate aerosol generation. The sample is thenhomogenised in a Mini-BeadBeater Type BX-4 (Biospec Products). Necroticfatty tissue is strain treated for 100 seconds at 5000 rpm in order toachieve bacterial lysis. In vivo grown bacteria require longer treatmentthan in vitro grown Staphylococcus aureus Staphylococcus which aredisrupted by a 30 second bead-beat.

After bead-beating the tubes are chilled on ice before opening in afume-hood as heat generated during disruption may degrade the TRIzol andrelease cyanide.

200 microliters of chloroform is then added and the tubes shaken by handfor 15 seconds to ensure complete mixing. After 2-3 minutes at roomtemperature the tubes are spun down at 12,000×g, 4° C. for 15 minutesand RNA extraction is then continued according to the method given bythe manufacturers of TRIzol Reagent i.e.:—The aqueous phase, approx 0.6ml, is transferred to a sterile eppendorf tube and 0.5 ml of isopropanolis added. After 10 minutes at room temperature the samples are spun at12,000×g, 4° C. for 10 minutes. The supernatant is removed and discardedthen the RNA pellet is washed with 1 ml 75% ethanol. A brief vortex isused to mix the sample before centrifuging at 7,500×g, 4° C. for 5minutes. The ethanol is removed and the RNA pellet dried under vacuumfor no more than 5 minutes. Samples are then resuspended by repeatedpipetting in 100 microliters of DEPC treated water, followed by 5-10minutes at 55° C. Finally, after at least 1 minute on ice, 200 units ofRnasin (Promega) is added.

RNA preparations are stored at −80° C. for up to one month. For longerterm storage the RNA precipitate can be stored at the wash stage of theprotocol in 75% ethanol for at least one year at −20° C.

Quality of the RNA isolated is assessed by running samples on 1% agarosegels. 1×TBE gels stained with ethidium bromide are used to visualisetotal RNA yields. To demonstrate the isolation of bacterial RNA from theinfected tissue 1×MOPS, 2.2M formaldehyde gels are run and vacuumblotted to Hybond-N (Amersham). The blot is then hybridised with a ³²Plabelled oligonucletide probe specific to 16s rRNA of Staphylococcusaureus ( K. Greisen, M. Loeffelholz, A. Purohit and D. Leong. J.Clin.(1994) Microbiol. 32 335-351 An oligonucleotide selected from Table 1 isused as a probe. The size of the hybridising band is compared to that ofcontrol RNA isolated from in vitro grown Staphylococcus aureus WCUH29 inthe Northern blot. Correct sized bacterial 16s rRNA bands can bedetected in total RNA samples which show extensive degradation of themammalian RNA when visualised on TBE gels.

c) The removal of DNA from Staphylococcus aureus WCUH29-derived RNA

DNA was removed from 73 microliter samples of RNA by a 15 minutetreatment on ice with 3 units of DNAaseI, amplification grade (GibcoBRL, Life Technologies) in the buffer supplied with the addition of 200units of Rnasin (Promega) in a final volume of 90 microliters.

The DNAase was inactivated and removed by treatment with TRIzol LSReagent (Gibco BRL, Life Technologies) according to the manufacturersprotocol. DNAase treated RNA was resuspended in 73 microliters of DEPCtreated water with the addition of Rnasin as described in Method 1.

d) The Preparation of cDNA from RNA Samples Derived from Infected Tissue

10 microliter samples of DNAase treated RNA are reverse transcribedusing SuperScript Preamplification System for First Strand cDNASynthesis kit (Gibco BRL, Life Technologies) according to themanufacturers instructions. 1 nanogram of random hexamers is used toprime each reaction. Controls without the addition of SuperScriptIIreverse transcriptase are also run. Both+/−RT samples are treated withRNaseH before proceeding to the PCR reaction

e) The Use of PCR to Determine the Presence of a Bacterial cDNA Species

PCR reactions are set up on ice in 0.2 ml tubes by adding the followingcomponents: 45 microliters PCR SUPERMIX (Gibco BRL, Life Technologies);1 microlitre 50 mM MgCl₂, to adjust final concentration to 2.5 mM; 1microlitre PCR primers(optimally 18-25 basepairs in length and designedto possess similar annealing temperatures), each primer at 10 mM initialconcentration; and 2 microlitres cDNA.

PCR reactions are run on a Perkin Elmer GeneAmp PCR System 9600 asfollows: 5 minutes at 95° C., then 50 cycles of 30 seconds each at 94°C., 42° C. and 72° C. followed by 3 minutes at 72° C. and then a holdtemperature of 4° C. (the number of cycles is optimally 30-50 todetermine the appearance or lack of a PCR product and optimally 8-30cycles if an estimation of the starting quantity of cDNA from the RTreaction is to be made); 10 microlitre aliquots are then run out on 1%1×TBE gels stained with ethidium bromide with PCR product, if present,sizes estimated by comparison to a 100 bp DNA Ladder (Gibco BRL, LifeTechnologies). Alternatively if the PCR products are convenientlylabelled by the use of a labelled PCR primer (e.g. labelled at the 5′end with a dye) a suitable aliquot of the PCR product is run out on apolyacrylamide sequencing gel and its presence and quantity detectedusing a suitable gel scanning system (e.g. ABI Prism™377 Sequencer usingGeneScan™ software as supplied by Perkin Elmer).

RT/PCR controls may include +/− reverse transcriptase reactions, 16srRNA primers or DNA specific primer pairs designed to produce PCRproducts from non-transcribed Staphylococcus aureus WCUH29 genomicsequences.

To test the efficiency of the primer pairs they are used in DNA PCR withStaphylococcus aureus WCUH29 total DNA. PCR reactions are set up and runas described above using approx. 1 microgram of DNA in place of the cDNAand 35 cycles of PCR.

Primer pairs which fail to give the predicted sized product in eitherDNA PCR or RT/PCR are PCR failures and as such are uninformative. Ofthose which give the correct size product with DNA PCR two classes aredistinguished in RT/PCR: 1.Genes which are not transcribed in vivoreproducibly fail to give a product in RT/PCR; and 2.Genes which aretranscribed in vivo reproducibly give the correct size product in RT/PCRand show a stronger signal in the +RT samples than the signal (if at allpresent) in -RT controls.

The polynucleotide sequence set forth in [SEQ ID NO: 1] was identifiedin the above test as being transcribed in vivo. Deduced amino acidsequence is given as SEQ ID NO:2. The pair of PCR primers used toidentify the gene are given as SEQ ID NOS:5 and 6. Primers of SEQ ID NO:3 and 4 may be similarly used.

8 2943 base pairs nucleic acid double linear Genomic DNA 1 CTGTTAGAAAAGAAGGGAAA TTACCTCGTG AAGTCATTCG TTATGAGTAT GACCTAGAAT 60 ATGGTACAAATGTTTTAACA ATGCACAAAG ATGCAATTAA ACCAGGTCAA CGTGTGTTAA 120 TTACAGATGATTTATTAGCT ACTGGTGGTA CGATTGAAGC AGCAATAAAA TTAGTTGAAA 180 AATTAGGCGGTATCGTAGTA GGTATTGCAT TTATAATTGA ATTGAAATAT TTAAATGGTA 240 TTGAAAAAATTAAAGATTAC GATGTTATGA GTTTAATCTC ATACGACGAA TAATAAATAA 300 TATAATTTTATCAAATGAAA TCCTTCATCA AATGTATAAG AACCAATGAC TTAATTAAAA 360 AAGTTGTTTAAGTTTTCTTA ACATGAGATG TTAGGATTTT TTATTTACTG AAAATGTTAG 420 ATGATTGAGCATTATACCTT AATAACATCG TTTATTTATT TCATAAATTG TAGTATCATA 480 GAACTAATATTTAAAAAATG AAACAGTAGA TTTAGGTCGA ATTTTTGTAA AAGTTTTAAA 540 AGTAGGAATAGTATACAAAT TAAACTCGCT CAAGTAAAAT TAATATTACG ATTAATGACG 600 ACAGGATAAATATTTATCGT CGACGGACGT ATGATTGGTG TGGGACAAAT ACTATTCAAC 660 AAGAGTACCTAAATCATTGT TTAAGGCGAA GTAATAAATA TGAATGGGGT GTATCATATA 720 ATGAACAACGAATATCCATA TAGTGCAGAT GAAGTTCTTC ACAAAGCAAA ATCATATTTG 780 TCAGCAGATGAATATGAGTA TGTTTTAAAA AGCTATCATA TTGCTTATGA AGCACATAAA 840 GGTCAGTTCCGAAAAAACGG ATTACCATAC ATTATGCATC CTATACAAGT TGCAGGTATT 900 TTAACAGAAATGCGATTAGA CGGACCGACG ATTGTCGCAG GTTTTTTGCA TGATGTAATT 960 GAAGATACACCGTATACATT TGAAGATGTA AAAGAAATGT TCAATGAAGA AGTTGCTCGA 1020 ATTGTTGATGGTGTGACGAA GCTTAAAAAA GTAAAATACC GCTCAAAAGA AGAACAACAA 1080 GCTGAAAATCATCGCAAGTT ATTTATTGCG ATTGCCAAAG ATGTACGCGT AATTTTGGTG 1140 AAATTAGCAGACAGATTACA TAATATGCGT ACCTTGAAAG CCATGCCGCG TGAAAAACAA 1200 ATTAGAATTTCTCGAGAAAC ATTAGAAATT TATGCACCTT TAGCACATCG TCTTGGTATT 1260 AATACAATTAAATGGGAACT AGAAGATACG GCTCTTCGTT ATATTGATAA TGTGCAATAT 1320 TTTAGAATAGTCAATTTAAT GAAGAAGAAA CGTAGTGAAC GTGAAGCGTA TATCGAAACG 1380 GCTATTGATAGAATACGTAC TGAAATGGAC CGAATGAATA TCGAAGGCGA TATAAATGGT 1440 AGACCTAAACATATTTACAG TATTTATCGG AAAATGATGA AGCAGAAAAA ACAATTTGAT 1500 CAAATTTTTGATTTGTTGGC GATACGTGTT ATTGTCAATT CTATTAATGA TTGTTATGCG 1560 ATACTTGGGTTGGTGCATAC GTTATGGAAA CCGATGCCAG GACGTTTTAA AGATTATATT 1620 GCAATGCCTAAACAAAATTT GTATCAGTCA TTGCATACTA CAGTAGTAGG TCCAAATGGA 1680 GACCCGCTCGAAATCCAAAT ACGAACGTTT GATATGCACG AAATTGCTGA GCATGGTGTT 1740 GCAGCACACTGGGCTTACAA AGAAGGTAAA AAAGTAAGTG AAAAAGATCA AACTTATCAA 1800 AATAAGTTAAATTGGTTAAA AGAATTAGCT GAAGCGGATC ATACATCGTC TGACGCTCAA 1860 GAATTTATGGAAACCTTAAA ATATGACTTA CAGAGTGACA AAGTATACGC ATTTACCCCA 1920 GCGAGTGATGTTATTGAGTT GCCATATGGT GCTGTGCCGA TTGATTTTGC TTATGCGATT 1980 CACAGTGAAGTAGGTAATAA GATGATTGGT GCCAAGGTGA ATGGCAAAAT TGTACCAATT 2040 GACTATATTTTACAAACAGG CGATATTGTT GAAATACGTA CTAGTAAACA TTCATATGGA 2100 CCAAGTCGTGATTGGTTGAA AATTGTTAAA TCGTCTAGTG CCAAAGGTAA AATTAAAAGT 2160 TTCTTCAAAAAACAAGATCG TTCATCTAAT ATTGAAAAAG GCCGAATGAT GGTTGAAGTT 2220 GAAATAAAAGAGCAAGGATT TAGAGTCGAA GATATTTTGA CAGAGAAAAA TATTCAGGTT 2280 GTTAATGAAAAATATAACTT TGCAAATGAA GATGATTTAT TCGCAGCTGT AGGATTTGGC 2340 GGCGTGACATCCTTACAGAT TGTTAATAAA TTAACTGAAA GACAACGTAT TTTAGATAAA 2400 CAACGTGCTTTAAATGAAGC ACAAGAAGTT ACGAAATCAT TGCCTATTAA AGACAACATC 2460 ATTACTGATAGTGGTGTCTA TGTAGAAGGT TTAGAAAATG TACTTATCAA GTTGTCAAAA 2520 TGTTGTAATCCTATACCAGG TGATGATATT GTAGGTTATA TCACCAAAGG TCACGGTATT 2580 AAAGTACATCGCACTGATTG CCCAAATATT AAGAACGAAA CTGAACGACT AATTAATGTT 2640 GAATGGGTAAAATCAAAAGA CGCAACTCAA AAATATCAGG TTGATTTAGA GGTAACTGCG 2700 TATGACCGAAATGGCTTGTT GAATGAAGTA CTACAAGCTG TTAGCTCGAC AGCCGGCAAT 2760 TTAATTAAAGTTTCAGGACG TTCAGATATT GATAAAAATG CAATAATAAA TATTAGTGTC 2820 ATGGTGAAAAACGTGAATGA TGTTTATCGT GTGGTAGAAA AGATCAAACA ACTTGGTGAT 2880 GTTTATACAGTAACAAGAGT TTGGAACTAG AGGTGCAAAA TATGAAAGTA GTTGTACAAA 2940 GAG 2943 736amino acids amino acid single linear peptide 2 Met Asn Gly Val Tyr HisIle Met Asn Asn Glu Tyr Pro Tyr Ser Ala 1 5 10 15 Asp Glu Val Leu HisLys Ala Lys Ser Tyr Leu Ser Ala Asp Glu Tyr 20 25 30 Glu Tyr Val Leu LysSer Tyr His Ile Ala Tyr Glu Ala His Lys Gly 35 40 45 Gln Phe Arg Lys AsnGly Leu Pro Tyr Ile Met His Pro Ile Gln Val 50 55 60 Ala Gly Ile Leu ThrGlu Met Arg Leu Asp Gly Pro Thr Ile Val Ala 65 70 75 80 Gly Phe Leu HisAsp Val Ile Glu Asp Thr Pro Tyr Thr Phe Glu Asp 85 90 95 Val Lys Glu MetPhe Asn Glu Glu Val Ala Arg Ile Val Asp Gly Val 100 105 110 Thr Lys LeuLys Lys Val Lys Tyr Arg Ser Lys Glu Glu Gln Gln Ala 115 120 125 Glu AsnHis Arg Lys Leu Phe Ile Ala Ile Ala Lys Asp Val Arg Val 130 135 140 IleLeu Val Lys Leu Ala Asp Arg Leu His Asn Met Arg Thr Leu Lys 145 150 155160 Ala Met Pro Arg Glu Lys Gln Ile Arg Ile Ser Arg Glu Thr Leu Glu 165170 175 Ile Tyr Ala Pro Leu Ala His Arg Leu Gly Ile Asn Thr Ile Lys Trp180 185 190 Glu Leu Glu Asp Thr Ala Leu Arg Tyr Ile Asp Asn Val Gln TyrPhe 195 200 205 Arg Ile Val Asn Leu Met Lys Lys Lys Arg Ser Glu Arg GluAla Tyr 210 215 220 Ile Glu Thr Ala Ile Asp Arg Ile Arg Thr Glu Met AspArg Met Asn 225 230 235 240 Ile Glu Gly Asp Ile Asn Gly Arg Pro Lys HisIle Tyr Ser Ile Tyr 245 250 255 Arg Lys Met Met Lys Gln Lys Lys Gln PheAsp Gln Ile Phe Asp Leu 260 265 270 Leu Ala Ile Arg Val Ile Val Asn SerIle Asn Asp Cys Tyr Ala Ile 275 280 285 Leu Gly Leu Val His Thr Leu TrpLys Pro Met Pro Gly Arg Phe Lys 290 295 300 Asp Tyr Ile Ala Met Pro LysGln Asn Leu Tyr Gln Ser Leu His Thr 305 310 315 320 Thr Val Val Gly ProAsn Gly Asp Pro Leu Glu Ile Gln Ile Arg Thr 325 330 335 Phe Asp Met HisGlu Ile Ala Glu His Gly Val Ala Ala His Trp Ala 340 345 350 Tyr Lys GluGly Lys Lys Val Ser Glu Lys Asp Gln Thr Tyr Gln Asn 355 360 365 Lys LeuAsn Trp Leu Lys Glu Leu Ala Glu Ala Asp His Thr Ser Ser 370 375 380 AspAla Gln Glu Phe Met Glu Thr Leu Lys Tyr Asp Leu Gln Ser Asp 385 390 395400 Lys Val Tyr Ala Phe Thr Pro Ala Ser Asp Val Ile Glu Leu Pro Tyr 405410 415 Gly Ala Val Pro Ile Asp Phe Ala Tyr Ala Ile His Ser Glu Val Gly420 425 430 Asn Lys Met Ile Gly Ala Lys Val Asn Gly Lys Ile Val Pro IleAsp 435 440 445 Tyr Ile Leu Gln Thr Gly Asp Ile Val Glu Ile Arg Thr SerLys His 450 455 460 Ser Tyr Gly Pro Ser Arg Asp Trp Leu Lys Ile Val LysSer Ser Ser 465 470 475 480 Ala Lys Gly Lys Ile Lys Ser Phe Phe Lys LysGln Asp Arg Ser Ser 485 490 495 Asn Ile Glu Lys Gly Arg Met Met Val GluVal Glu Ile Lys Glu Gln 500 505 510 Gly Phe Arg Val Glu Asp Ile Leu ThrGlu Lys Asn Ile Gln Val Val 515 520 525 Asn Glu Lys Tyr Asn Phe Ala AsnGlu Asp Asp Leu Phe Ala Ala Val 530 535 540 Gly Phe Gly Gly Val Thr SerLeu Gln Ile Val Asn Lys Leu Thr Glu 545 550 555 560 Arg Gln Arg Ile LeuAsp Lys Gln Arg Ala Leu Asn Glu Ala Gln Glu 565 570 575 Val Thr Lys SerLeu Pro Ile Lys Asp Asn Ile Ile Thr Asp Ser Gly 580 585 590 Val Tyr ValGlu Gly Leu Glu Asn Val Leu Ile Lys Leu Ser Lys Cys 595 600 605 Cys AsnPro Ile Pro Gly Asp Asp Ile Val Gly Tyr Ile Thr Lys Gly 610 615 620 HisGly Ile Lys Val His Arg Thr Asp Cys Pro Asn Ile Lys Asn Glu 625 630 635640 Thr Glu Arg Leu Ile Asn Val Glu Trp Val Lys Ser Lys Asp Ala Thr 645650 655 Gln Lys Tyr Gln Val Asp Leu Glu Val Thr Ala Tyr Asp Arg Asn Gly660 665 670 Leu Leu Asn Glu Val Leu Gln Ala Val Ser Ser Thr Ala Gly AsnLeu 675 680 685 Ile Lys Val Ser Gly Arg Ser Asp Ile Asp Lys Asn Ala IleIle Asn 690 695 700 Ile Ser Val Met Val Lys Asn Val Asn Asp Val Tyr ArgVal Val Glu 705 710 715 720 Lys Ile Lys Gln Leu Gly Asp Val Tyr Thr ValThr Arg Val Trp Asn 725 730 735 32 base pairs nucleic acid single linearGenomic DNA 3 CCGCCATGGG TAACAACGAA TATCCATATA GT 32 29 base pairsnucleic acid single linear Genomic DNA 4 CGGGATCCGC ACCTCTAGTT CCAAACTCT29 18 base pairs nucleic acid single linear Genomic DNA 5 AGATACGTACTGAAATGG 18 18 base pairs nucleic acid single linear Genomic DNA 6CCTGTGATTC GCATAAGC 18 809 base pairs nucleic acid double linear GenomicDNA 7 CGGCTCTTCG TAATATTGAT AATGTGCAAT ATTTNAAGAA TAATCAATTT ATTGAAGAAG60 AAACCGTAGT GACCGTGAGC GAATATCGAA NCGGCTATTG ATAGAATACG TACTGAAATG 120GACCCGAATG AATATCGAAG NCGATATAAA TGGTAGACCT AAACATATTT ACAGTATTTA 180TCGGNAAATG ATGAAGCAGA AAAAACAATT TGATCAAATT TTTGATTTGT TGGCGATACG 240TGTTATTGTC AATTCTATTA ATGATTGTTA TGCGATACTT GGGTTGGTGC ATACGTTATG 300GAAACCGATG CCAGGACGTT TTAAAGATTA TATTGCAATG CCTAAACAAA ATTTGTATCA 360GTCATTGCAT ACTACAGTAG TAGGTCCAAA TGGAGACCCG CTCGAAATCC AAATACGAAC 420GTTTGATATG CACGAAATTG CTGAGCATGG TGTTGCAGCA CACTGGGCTT ACAAAGAAGG 480TAAAAAAGTA AGTGAAAAAG ATCAAACTTA TCAAAATAAG TTAAATTGGT TAAAAGAATT 540AGCTGAAGCG GATCATACAT CGTCTGACGC TCAAGAATTT ATGGAAACCT TATAATATGA 600CTTACAGAGT GACAAAGTAT ACGCATTTAC CCCAGGGAGT GATGTTATTG AGTNGGCATA 660TGGTGCTGTG CCGATTGGAT TTTGGCTTAT GCGAATCACA GGGAANGTAG GTAATAAGAT 720GATTGGCGCC CAGGTGGAAT GGCAAAATTG TACCANATTG ACTTATNTTT TCACAAAACA 780GGCGGATATT GTTGGAAATA CCGTTCTAG 809 155 amino acids amino acid singlelinear peptide 8 Met Asn Ile Glu Xaa Asp Ile Asn Gly Arg Pro Lys His IleTyr Ser 1 5 10 15 Ile Tyr Arg Xaa Met Met Lys Gln Lys Lys Gln Phe AspGln Ile Phe 20 25 30 Asp Leu Leu Ala Ile Arg Val Ile Val Asn Ser Ile AsnAsp Cys Tyr 35 40 45 Ala Ile Leu Gly Leu Val His Thr Leu Trp Lys Pro MetPro Gly Arg 50 55 60 Phe Lys Asp Tyr Ile Ala Met Pro Lys Gln Asn Leu TyrGln Ser Leu 65 70 75 80 His Thr Thr Val Val Gly Pro Asn Gly Asp Pro LeuGlu Ile Gln Ile 85 90 95 Arg Thr Phe Asp Met His Glu Ile Ala Glu His GlyVal Ala Ala His 100 105 110 Trp Ala Tyr Lys Glu Gly Lys Lys Val Ser GluLys Asp Gln Thr Tyr 115 120 125 Gln Asn Lys Leu Asn Trp Leu Lys Glu LeuAla Glu Ala Asp His Thr 130 135 140 Ser Ser Asp Ala Gln Glu Phe Met GluThr Leu 145 150 155

What is claimed is:
 1. An isolated polypeptide comprising SEQ ID NO:2.2. A composition comprising the isolated polypeptide of claim 1 and acarrier.
 3. The isolated polypeptide of claim 1, wherein the isolatedpolypeptide further comprises a heterologous amino acid sequence.
 4. Acomposition comprising the isolated polypeptide of claim 3 and acarrier.
 5. The isolated polypeptide of claim 1, wherein the isolatedpolypeptide consists of SEQ ID NO:2.
 6. A composition comprising theisolated polypeptide of claim 5 and a carrier.